Double-antibody sandwich ELISA (enzyme linked immuno-sorbent assay) test method for ovalbumin
A technology of ovalbumin and a detection method, which is applied in the field of biological inspection and quarantine, can solve the problems of inability to process evaluation and high price, and achieve the effects of simple and convenient detection procedures, time saving and short detection time.
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Embodiment 1
[0032] Example 1 Immune BALB / C mice with highly purified ovalbumin
[0033] The specific immunization method is as follows:
[0034]Healthy BALB / C female mice were immunized with two immunization schemes, one was footpad rapid immunization scheme: 100 μL of 1 mg / mL OVA was mixed with the same amount of Freund's complete adjuvant, and emulsified by an emulsifier for 40 minutes. After three BALB / C mice were anesthetized with ether, the two hind paws of the mice were disinfected with alcohol cotton and injected subcutaneously, with 30 μL of immunogen injected into each hind paw. Seven days after the first immunization, mix 100 μL of 1 mg / mL BALB / C with the same amount of Freund’s incomplete adjuvant, and then inject it in the same way as above. 14 days after the first immunization, blood was collected from the tail to detect the serum titer of the mice. The other is the routine immunization scheme: the first immunoemulsification method is the same as the foot pad rapid immunizat...
Embodiment
[0035] Example 2 Cell fusion and screening and cloning of positive hybridoma cell lines
[0036] The specific operation steps are as follows:
[0037] Blood was collected from the eyeballs of the mice 3 days after the strong immunization, and the serum was separated as a positive control. The spleen of the mice was aseptically removed (the popliteal lymph nodes were aseptically removed from the footpad immunized mice), and the spleen was repeatedly washed with 1640 medium with a syringe to prepare splenocytes. Suspension (or use a syringe to grind popliteal lymph nodes to prepare cell suspension), fully mix with SP2 / 0 cells at a ratio of 1: (5-10), perform cell fusion with 50% PEG1000 in a water bath at 37°C, and fused Add the cell suspension to the HAT-1640 culture medium containing 20% fetal bovine serum, add it to the 96-well cell culture plate with feeder cells in advance, 100ul per well, put it in 37℃, 5%CO 2 Culture in an incubator, and on the 4th, 7th, and 10th day, ...
Embodiment 3
[0038] Example 3 Identification of monoclonal antibody subclasses and large-scale preparation of ascites
[0039] The specific method is as follows:
[0040] (1) Coat the microtiter plate with OVA for detection of proovalbumin, overnight at 4°C;
[0041] (2) Wash with PBST 3 times and spin dry;
[0042] (3) Add appropriately diluted ascites or cell culture supernatant, 37°C, 1h;
[0043] (4) Wash 3 times with PBST, add monoclonal antibody subclass identification reagent, 37°C, 1h;
[0044] (5) Wash 3 times with PBST and spin dry;
[0045] (6) Add 100ul of substrate solution to each well, develop color at 37°C for 15min, and read the value on a microplate reader to judge the positive result.
[0046] Monoclonal Antibody Subclass Identification
[0047]
[0048] The specific operation of in vitro large-scale induction of ascites: take 8-10 week-old robust BALB / C mice, and inject 0.5ml of high-pressure sterilized paraffin oil into each mouse intraperitoneally, and after 1...
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